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Background The schistosome egg represents an attractive developmental stage at which to target transgenes because of the high ratio of germ to somatic cells, because the transgene might be propagated and amplified by infecting snails with the miracidia hatched from treated eggs, and because eggs can be readily obtained from experimentally infected rodents. Methods/Findings We investigated the utility of square wave electroporation to deliver transgenes and other macromolecules including fluorescent (Cy3) short interference (si) RNA molecules, messenger RNAs, and virions into eggs of Schistosoma mansoni. First, eggs were incubated in Cy3-labeled siRNA with and without square wave electroporation. Cy3-signals were detected by fluorescence microscopy in eggs and miracidia hatched from treated eggs. Second, electroporation was employed to introduce mRNA encoding firefly luciferase into eggs. Luciferase activity was detected three hours later, whereas luciferase was not evident in eggs soaked in the mRNA. Third, schistosome eggs were exposed to Moloney murine leukemia virus virions (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG). Proviral transgenes were detected by PCR in genomic DNA from miracidia hatched from virion-exposed eggs, indicating the presence of transgenes in larval schistosomes that had been either soaked or electroporated. However, quantitative PCR (qPCR) analysis determined that electroporation of virions resulted in 2–3 times as many copies of provirus in these schistosomes compared to soaking alone. In addition, relative qPCR indicated a copy number for the proviral luciferase transgene of ∼20 copies for 100 copies of a representative single copy endogenous gene (encoding cathepsin D). Conclusions Square wave electroporation facilitates introduction of transgenes into the schistosome egg. Electroporation was more effective for the transduction of eggs with pseudotyped MLV than simply soaking the eggs in virions. These findings underscore the potential of targeting the schistosome egg for germ line transgenesis., Author Summary The genome sequences of two of the three major species of schistosomes are now available. Molecular tools are needed to determine the importance of these new genes. With this in mind, we investigated introduction of reporter transgenes into schistosome eggs, with the longer-term aim of manipulation of schistosome genes and gene functions. The egg is a desirable developmental stage for genome manipulation, not least because it contains apparently accessible germ cells. Introduction of transgenes into the germ cells of schistosome eggs might result in transgenic schistosomes. However, the egg is surrounded by a thick shell which might block access to entry of transgenes. We cultured eggs in the presence of three types of reporter transgenes of increasing molecular size, and in addition we tried to produce transient holes in the eggs by electroporation to investigate whether the transgenes would more easily enter the eggs. Electroporation of eggs appeared to allow entry of two larger types of transgenes into cultured schistosome eggs, messenger RNA encoding firefly luciferase, and retroviral virions. We anticipate that this approach, electroporation of transgenes into schistosome eggs, will facilitate genetic manipulation of schistosomes for investigating the importance of schistosome genes and gene products as new intervention targets.

Background The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. Methodology/Principal Findings Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt) encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa), prosegment (95 aa), and mature protease (213 aa). BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%), Paragonimus westermani (58%), Schistosoma mansoni and S. japonicum (52%), and with vertebrate cathepsin F (51%). Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of ∼3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and activate to a mature enzyme, trans-processing by Fasciola hepatica cathepsin L cleaved the prosegment of Ov-CF-1, releasing a mature cathepsin F with activity against the peptide Z-Phe-Arg-NHMec >50 times that of the zymogen. Immunocytochemistry using antibodies raised against the recombinant enzyme showed that Ov-CF-1 is expressed in the gut of the mature hermaphroditic fluke and also in the reproductive structures, including vitelline glands, egg, and testis. Ov-CF-1 was detected in bile duct epithelial cells surrounding the flukes several weeks after infection of hamsters with O. viverrini and, in addition, had accumulated in the secondary (small) bile ducts where flukes cannot reach due to their large size. Conclusions/Significance A cathepsin F cysteine protease of the human liver fluke O. viverrini has been characterized at the gene and protein level. Secretion of this protease may contribute to the hepatobiliary abnormalities, including cholangiocarcinogenesis, observed in individuals infected with this parasite., Author Summary Opisthorchiasis, oriental liver fluke infection, is a food-borne parasitic disease that afflicts millions of residents in northern Thailand and Laos. Related infections occur in North Asia, including China and Korea. This kind of liver fluke infection is the consequence of eating certain uncooked or undercooked freshwater fish contaminated with the larvae of the parasite Opisthorchis viverrini. Whereas the infection can cause disease in the bile ducts and liver, infection with the oriental fluke can lead to the development of a liver cancer, cholangiocarcinoma (bile duct cancer). Our recent studies have begun to focus on products and metabolites from the parasite that are carcinogenic. Many proteolytic enzymes are known to be secreted by parasites. This report centers on a specific category of protease, termed cathepsin F. We determined here that O. viverrini expresses a cathepsin F in its gut and in other organs. In the liver fluke, cathepsin F likely plays a role in digesting ingested human cells. The gene encoding the parasite enzyme shows evolutionary relatedness to a similar gene in humans. The fluke cathepsin F also is released from the parasite into livers of infected mammals, where it appears to contribute to inflammation surrounding the parasite. In this regard, it may be involved in early events that lead to bile duct cancer.

The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis., Author Summary Reverse genetics tools allow assessing the function of unknown genes. Their application for the study of neglected infectious diseases could lead eventually to the identification of relevant gene products to be used in diagnosis, or as drug targets or immunization candidates. Being technically more simple and less demanding than other reverse genetics tools such as transgenesis or knockouts, the suppression of gene activity mediated by double-stranded RNA has emerged as a powerful tool for the analysis of gene function. RNAi appeared as an obvious alternative to apply in complex biological systems where information is still scarce, a situation common to several infectious and parasitic diseases. However, several technical or practical difficulties have hampered the development of this technique in parasites to the expectations originally generated. We developed a simple method to test the presence of a viable RNAi pathway by silencing an exogenous reporter gene. The method was tested in F. hepatica, describing the conditions for transfection and confirming the existence of a viable RNAi pathway in this parasite. The experimental design created can be useful as a first approach in organisms where genetic analysis is still unavailable, providing a tool to unravel gene function and probably advancing new candidates relevant in pathobiology, prevention or treatment.